-Adrenoceptors (-ARs) are seven transmembrane domain, G-protein coupled receptors that transduce the cellular effects of epinephrine and norepinephrine and play a pivotal role in the vertebrate stress response. This study reports the cloning and characterization of two previously unreported -ARs from the rainbow trout (Oncorhynchus mykiss). Phylogenetic analysis of amino acid sequences indicates that both -ARs are homologs of the mammalian ₃-AR. Analysis of tissue expression patterns indicates that one of these trout ₃-adrenoceptors (_3a-AR) is highly expressed in gill and heart while the second (_3b-AR) is highly expressed by red blood cells (RBC). Expression of the _3b-AR in the red cell coupled with the finding of a single category of -AR binding sites on red cell membranes provides strong evidence for the control of the trout red cell -AR Na⁺/H⁺ exchanger (NHE) activity by signaling through this _3b-subtype and not through a ₁-subtype as previously proposed. The red cell specific trout _3b-AR exhibits binding characteristics that distinguish this receptor from each of the three pharmacologically defined categories of mammalian -ARs (₁-, ₂-, and ₃-AR). This study is the first to report the presence of a ₃-AR subtype in a fish species and the proposal that the _3b-AR controls red blood cell NHE activity represents a novel role for the ₃-AR subtype in vertebrates.